1. Field of the Invention
The present invention relates to a novel plasmid and use thereof. More particularly, the present invention relates to a plasmid capable of efficient expression of tumor necrosis factor (hereunder abbreviated as TNF) which is a protein having anti-tumor activity, a process for producing TNF using a host transformed with said plasmid, and a composition containing TNF produced by said process.
2. Prior Art
In 1975, Carswell and his co-workers found a substance having tumor cell killing or tumor necrotic activity in sera from animals previously sensitized with an immunopotentiator, and they referred to the substance as TNF (Proc. Natl. Acad. Sci. US 72: 3666-3670, 1975). It was later found that TNF caused hemorrhagic necrosis of various tumors with no apparent effect on the host and displayed cytocidal and cytostatic activities against several transformed cells (those which differentiated to tumors) in vitro without affecting normal cells. Because of these features, TNF holds great promise as an anti-tumor or cancer control agent. Ruff, H. R. & Gifford, G. E. reported in Lymphokines, vol. 2 (ed. Pick, E), 235-272, Academic Press, New York, 1981 that TNF was produced from activated macrophages in the organism. A substance having TNF activity was also isolated from the liquid culture of an established macrophage-like cell line. Most recently, two groups of researchers, with the aid of recombinant DNA techniques, revealed the amino acid sequence of human TNF (protein) produced by activated human macrophage-like cells (Pennica, D. et al.; Nature 312: 724-729, 1984; and Wang, A. M. et al.; Science 228: 149-154, 1985). In the study carried out by each group, mRNA of human TNF was isolated from activated human macrophage-like cells HL-60 and a cDNA library prepared from said mRNA was cloned for determination of the base sequence of TNF. At the same time, they purified TNF from the liquid culture of HL-60 and determined the amino terminal amino acids. As a result, they concluded that mature human TNF was a polypeptide composed of a 157-amino acid sequence starting with Val-Arg-Ser . . . and ending with Leu at the carboxyl terminus, as shown in FIG. 1, and that a TNF precursor was a protein wherein an additional polypeptide composed of 76 amino acids was attached to the amino terminal end of the 157-amino acid composed polypeptide. They also succeeded in producing said human TNF polypeptide in transformed E. coli. Pennica, D. et al. (ibid.) prepared a plasmid having said mature TNF gene inserted downstream from the E. coli tryptophan gene promoter, operator and ribosome-binding (Shine-Dalgarno: SD) sequence, and, using this plasmid as a vector, produced TNF in E. coli (W3110) displaying a cytocidal activity against L-929 of approximately 3.times.10.sup.5 units per ml of culture at A.sub.550 =1, which corresponded to approximately 3.times.10.sup.5 molecules of active TNF per cell (assuming a specific activity of 10.sup.8 units per mg of protein). Wang, A. M. et al (ibid.) prepared a ColE1 derived plasmid having mature TNF gene inserted downstream of the bacteriophage .lambda.PL promoter and the .lambda. gene N ribosome binding sequence. This plasmid contained mutations conferring a temperature-sensitive Cop.sup.- phenotype (Wang, E. M. et al., Proc. Natl. Acad. Sci. USA 79: 3570, 1982). The E. coli strain DG95 harboring this plasmid was cultured and extracts of induced cultured had 2.3.times.10.sup.5 units of TNF activity per milliliter, with TNF protein representing about 8% of the total E. coli cellular protein (L-929 cells were used in TNF activity assay as in the study by Pennica et al. shown above).